title: Metagenomics for NoV surveillance: Test Set Sample sequencing
description: For Norovirus (NoV) genomic surveillance, metagenomics sequencing of viral nucleotides captured by VirCapSeq-VERT probe set, was performed. This metagenomics approach, in theory agnostic, offers the great advantage of unbiased detection of NoV, including the detection of novel NoV recombinants. Samples come from three different sequencing sets - Baseline Survey (BLS) samples, Outbreak-related (OB) samples, and two synthetic Test Set samples (TSS1 and TSS2). The TSS2 samples were prepared by oysters' bio-accumulation with selected human stool samples positive for NoV. The digestive tissues of the oysters were selected because it is known to concentrate most of the NoV contamination and is used in the standardized method for NoV and hepatitis A virus detection in shellfish (ISO 15216-1 2017). Digestive tissues from the laboratory-contaminated oysters were serially diluted in digestive tissues from oysters confirmed to be non-contaminated by norovirus, to obtain oyster samples with four levels of contamination: high, intermediate, medium-low and very-low. For the preparation of TSS2, in addition to the selection criterion for TSS1, the quantity of human stool sample available was a limiting factor. Indeed, for artificial contamination of oysters, the in-house protocol used around 10^7 NoV genome copies per strain, which ensures that oysters are efficiently and homogeneously contaminated, taking into account the loss of virus binding on oyster shells and tank walls (Maalouf et al., 2011). TSS2 contains 6 NoV strains, namely GII.17[P17], GII.3[P21], GII.4[P31], GII.2[P16], GI.4[P4], GI.1[P1], i.e. 4 strains belonging to GII and 2 belonging to GI. Strains belonging to NoV GII.4[P16], GII.4[P31] and GII.17[P17] genotypes were selected in priority due to their high frequency in the European human population since 2016 (Noronet unpublished data, search date 19-01-2019) (https://www.erasmusmc.nl/en/research/projects/Noronet).